Effects of nutrient supply on leaf stoichiometry and relative growth rate of three stoloniferous alien plants

Different nutrient supply brings about changes in leaf stoichiometry, which may affect growth rate and primary production of plants. Invasion of alien plants is a severe threat to biodiversity and ecosystem worldwide. A pot experiment was conducted by using three stoloniferous alien plants Wedelia trilobata, Alternanther philoxeroides and Hydrocotyle vulgaris to investigate effects of nutrient supply on their leaf stoichiometry and relative growth rate. Different nitrogen or phosphorus supply was applied in the experiment (N1:1 mmol L-1, N2:4 mmol L-1, and N3:8 mmol L-1, P1:0.15 mmol L-1, P2:0.6 mmol L-1 and P3:1.2 mmol L-1). Nitrogen and phosphorus concentrations in leaves of the three alien plants significantly increased with increase of nitrogen supply. With increase of phosphorus supply, nitrogen or phosphorus concentration of leaf was complex among the three alien plants. N:P ratio in leaf of the three alien plants subjected to different levels of nutrient supply was various. A positive correlation between relative growth rate and N:P ratio of the leaf is observed in W. trilobata and A. philoxeroides suffering from N-limitation. A similar pattern was not observed in Hydrocotyle vulgaris. We tentatively concluded that correlations between relative growth rate and N: P ratio of the leaf could be affected by species as well as nutrient supply. It is suggested that human activities, invasive history, local abundance of species et al maybe play an important role in the invasion of alien plants as well as relative growth rate.


Introduction
N:P ratio is a critical indicator of nutrient limitation (N vs P) in the terrestrial ecosystem [1]. Leaf stoichiometry can reflect nutrient allocation strategy, growth strategy and expanding ability of invasive plants [2]. Plant stoichiometry refers to balance and ratio of C, N, P in its issue or organ [3]. Primary production and litter decomposition of plants were significantly affected by C: N: P ratio of the leaf as well as community dynamics and nutrient cycling [4][5][6][7][8][9]. In addition, C: N: P ratio of the leaf was related to plant adaptation to specific environments [10,11]. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 The stolon of Alternanther philoxeroides usually takes root when in contact with moist substratum, forming a network of stolon [44]. A. philoxeroides is distributed in many areas of the world such as Australia, the United States, and China [45,46].
Each node along the stolon of Hydrocotyle vulgaris has the potential to produce an independent ramet that consists of a simple leaf and adventitious roots [47]. H. vulgaris is widely distributed in tropical and temperate regions such as Southeast Asia, Europe and North America.
In 2018, 10 original plants of W. trilobata were collected from Zhejiang Province, China. 20 original plants of A. philoxeroides and H. vulgaris were collected in Sichuan Province, China. All original plants were planted in a greenhouse located at Sichuan Normal University. The average temperature is about 25 ± 5˚C in the greenhouse.

Experimental design
In September 2019, stolon internodes of the three alien plants without leaves were chosen. The stolon internodes were about 3-5cm length in size. Balance was used to measure their fresh weight. According to the ratio of dry to fresh weight, we could obtain initial biomass of each stolon internode [19]. The stolon internodes were respectively grown in a plastic pot (12 cm in upper edge diameter, 9 cm in bottom diameter and 10.5 cm in height) filled with perlite. Same volume of nutrient solution was added to each plastic pot every two days.
At the end of the experiment, fully expanded and mature leaves were selected from regenerated clonal fragments and dried to constant weight. The leaf samples were powdered and sifted through 100-mesh. Mortar Autoanalyzer 3 system (Seal analytical, Germany) was used to measure the nitrogen and phosphorus concentrations of the leaf [48].
Finally, whole regenerated clonal fragments were harvested and dried to constant weight. Balance was used to determine their biomass. The relative growth rate was calculated according to following formula: In the experiment, M 0 is the initial biomass of the stolon internode, M t is the final biomass of regenerated clonal fragment, and t is duration of the experiment.
We estimate the balance of plant homeostasis by stoichiometric regulation coefficient H. The stoichiometric regulation coefficient H was calculated according to the following formula: In the experiment, x is N:P or C: N or C:P ratio of nutrition, y is N:P or C: N or C:P ratio of leaf, c is a constant [49].1/H is a slope value and it should be between 0 and 1. The classifications of H values are as follows: H>4(homeostasis), 4>H>2(weak homeostasis), 2>H>1.33 (weakly sensitive state), 1.33>H(sensitive state). But it is often understood as absolute homeostasis when the equation fitting the data is insignificant.

Statistical analyses
Two-way analysis of variance was performed to assess the effects of nitrogen, phosphorus supply and their interaction on leaf stoichiometry of the three alien plants. Linear regression was performed to explore the correlation between relative growth rate of the three alien plants and their leaf stoichiometry respectively. All analyses were conducted with SPSS 20.0 software (SPSS Inc).

Results
Nitrogen and phosphorus concentrations in the leaf of W. trilobata and H. vulgaris were significantly affected by nitrogen supply as well as interaction between nitrogen and phosphorus supply (Tables 2 and 3). Phosphorus concentration in the leaf of W. trilobata was significantly affected by phosphorus supply (Table 2). Nitrogen and phosphorus concentrations in the leaf of A. philoxeroides was significantly affected by nitrogen supply, phosphorus supply as well as interaction between nitrogen and phosphorus supply (Table 4).
With increase of nitrogen supply, nitrogen and phosphorus concentrations in the leaf of W. trilobata significantly increased (Figs 1A and 2A). Nitrogen concentration in the leaf of H. vulgaris significantly increased ( Fig 1B). Phosphorus concentration in the leaf of H. vulgaris significantly increased (P2 or P3). Phosphorus concentration in the leaf of H. vulgaris was Table 2. Effects (F value) of nitrogen and phosphorus supply and their interaction on the relative growth rate of W. trilobata, nitrogen and phosphorus concentrations, N: P ratio in its leaf.

Factors
Relative growth rate (g day -1 ) significantly higher in the treatment (N2P1) than in the treatment (N1P1). Then, phosphorus concentration in the leaf of H. vulgaris was significantly lower in the treatment (N3P1) than in the treatment (N2P1) (Fig 2B). Nitrogen concentration in the leaf of A. philoxeroides was significantly higher in treatment (N2P2) than in treatment (N1P2). Then, nitrogen concentration in the leaf of A. philoxeroides was significantly lower in the treatment (N3P2) than in the treatment (N2P2) (Fig 1C). Phosphorus concentration in the leaf of A. philoxeroides significantly increased ( Fig 2C).
With increase of phosphorus supply, phosphorus concentration in the leaf of W. trilobata significantly increased (Fig 2A). With increase of phosphorus supply, nitrogen concentration in the leaf significantly decreased when A. philoxeroides was subjected to nitrogen supply (N1 or N2). Nitrogen concentration in the leaf of A. philoxeroide was significantly lower in the treatment (N3P2) than in the treatment (N3P1). Then, nitrogen concentration in the leaf of A. philoxeroide was significantly higher in the treatment (N3P3) than in the treatment (N3P2) (Fig 1C). Phosphorus concentration in the leaf of A. philoxeroide was significantly lower in the treatment (N3P1) than in the treatment (N3P2). Then, phosphorus concentration in the leaf of A. philoxeroide was significantly higher in the treatment (N3P2) than in the treatment (N3P3). With increase of phosphorus supply, phosphorus concentration in the leaf significantly decreased when A. philoxeroides was subjected to nitrogen supply (N2) (Fig 2C).
N:P ratio in leaf of W. trilobata or A. philoxeroides was significantly affected by nitrogen, phosphorus supply and interaction between nitrogen and phosphorus supply (Tables 2 and 4). With increase of nitrogen supply, N:P ratio in leaf significantly increased when W. trilobata was subjected to phosphorus supply (P1 or P2). N:P ratio of leaf was significantly lower in the treatment (N1P3) than in the treatment (N2P3). Then, N:P ratio of leaf was significantly higher in the treatment (N2P3) than in the treatment (N3P3). With increase of phosphorus supply, N: P ratio in leaf significantly decreased when W. trilobata was subjected to nitrogen supply (N1 or N3). N:P ratio of leaf was significantly lower in the treatment (N2P1) than in the treatment Table 3

Factors
Relative growth rate (g day -1 )
With increase of nitrogen supply, N:P ratio in leaf significantly increased when H. vulgaris was subjected to the same level of phosphorus supply (P1, P2, P3). With increase of phosphorus supply, N:P ratio in leaf significantly decreased when H. vulgaris was subjected to nitrogen supply (N2 or N3). N:P ratio of leaf was significantly higher in the treatment (N1P2) than in the treatment (N1P1). Then, N:P ratio of leaf was significantly lower in the treatment (N1P3) than in the treatment (N1P2) (Fig 3B).
With increase of nitrogen supply, N:P ratio in leaf significantly decreased when A. philoxeroides was subjected to phosphorus supply (P3). N:P ratio of leaf was significantly lower in the treatment (N1P1) than in the treatment (N2P1). Then, N:P ratio of leaf was significantly higher in the treatment (N2P1) than in the treatment (N3P1). N:P ratio of leaf was significantly lower in the treatment (N1P2) than in the treatment (N2P2). Then, N:P ratio of leaf was significantly higher in the treatment (N2P2) than in the treatment (N3P2). With increase of phosphorus supply, N:P ratio in leaf significantly decreased when A. philoxeroides was subjected to nitrogen supply (N2). N:P ratio of leaf was significantly lower in the treatment (N1P2) than in the treatment (N1P1). Then, N:P ratio of leaf was significantly higher in the treatment (N1P3) than in the treatment (N1P2). N:P ratio of leaf was significantly lower in the treatment (N3P2) than in the treatment (N3P1). Then, N:P ratio of leaf was significantly higher in the treatment (N3P3) than in the treatment (N3P2) (Fig 3C).

PLOS ONE
Effects of nutrient supply on leaf stoichiometry and relative growth rate of three stoloniferous alien plants H (N:P) of W. trilobata and H. vulgaris was 2.78 and 6.25 respectively. The relationship between N:P ratio of leaf and N:P ratio in nutrition solution was not established in A. philoxeroide (Table 5). N:P ratio in leaf of W. trilobata and A. philoxeroides was positively correlated with their relative growth rate (Fig 4A and 4C). However, a similar correlation was not observed in H. vulgaris (Fig 4B).

Discussion
Nitrogen or phosphorus concentration in leaf of the three alien plants significantly increased with increase of nitrogen supply, which is consistent with previous studies [50,51]. On the one hand, nitrogen addition increased the nitrogen concentration of leaf [52][53][54][55]. On the other hand, nitrogen addition exerted a positive effect on phosphatase activity, which enhances phosphorus acquisition of plants grown in high phosphorus availability of soil [56][57][58].
Nitrogen concentration in the leaf of W. trilobata and H. vulgaris was not significantly affected by phosphorus supply, which is consistent with the previous study [59]. With increase

PLOS ONE
Effects of nutrient supply on leaf stoichiometry and relative growth rate of three stoloniferous alien plants of phosphorus supply, a tendency of decreasing was observed in the nitrogen concentration of leaf when A. philoxeroides was subjected to nitrogen supply (N1 or N2). The possible explanation is that phosphorus addition incurred the use of internal nitrogen in issue or organ of plant [18]. As the same time, the tendency of decreasing first and then increasing was observed in the nitrogen concentration of leaf when A. philoxeroides was subjected to nitrogen supply (N3). The possible explanation is that efficiency and proficiency of N resorption significantly   60]. The possible explanation is that the three alien plants are suffering from Nlimitation in the experiment [16,29,61]. N:P ratio in leaf was various when the three alien plants were subjected to different levels of nutrient supply. So, more studies are needed to explore the relationship between N:P ratio and nutrient supply.
In the experiment, relative growth rates of W. trilobata, A. philoxeroides and H. vulgaris were 0.065±0.008, 0.064±0.009 and 0.077±0.007 respectively. Based on their H (N:P) , W. trilobata, A. philoxeroides and H. vulgaris were classified as weak homeostasis, absolute homeostasis and homeostasis respectively [62]. Stoichiometric homeostasis presented a positive influence on relative growth rate of plants growing in different nutrient availability [49]. Species dominance in Inner Mongolia grassland was positively correlated with their stoichiometric homeostasis [63,64]. So, further studies are needed to explore the relationship between stoichiometric homeostasis and growth performance of the plant.
Relationship between relative growth rate of plants and the leaf N: P ratio of its leaf was affected by nutrient limitation [16,28]. With nitrogen addition, a positive correlation between relative growth rate and N: P ratio of the leaf was observed in Arabidopsis thaliana suffering from N-limitation [16]. Negative correlation between relative growth rate and N: P ratio of the leaf was observed in N. tangutorum [49]. Correlations between relative growth rate and N: P ratio of leaf were different among W. trilobata, A. philoxeroides and H. vulgaris. We tentatively concluded that correlations between relative growth rate and N: P ratio of the leaf could be affected by species as well as nutrient supply.
W  [65]. In addition, a high relative growth rate could partly explain the successful invasion of alien plants [66]. A similar pattern was not observed in the experiment. It is suggested that human activities, invasive history, local abundance of species et al maybe play an important role in invasion of alien plants as well as relative growth rate.